The aim of this competing continuation proposal is to develop a rational approach for metabolic engineering of secondary metabolite production using microbial genomic technologies. In this work both subset and genome-wide microarray methods will be used to analyze secondary metabolism in Streptomyces coelicolor. Quantitative physiological and modeling approaches will be combined to obtain information on temporal and conditional expression of global and pathway-specific regulatory factors for antibiotic biosynthetic pathways. In order to dissect further the control architecture in these multi-step biosynthetic systems, key regulatory elements will be investigated and their role in the circuitry of secondary metabolism defined. In addition, controlled expression of structural and regulatory genes in the actinorhodin and undecylprodigiosin biosynthesis will be analyzed to provide a genome-wide understanding of the intricate mechanisms affecting these secondary metabolic pathways. The specific objectives of this project are: I. Perform genome-wide microarray analysis to monitor expression of absA, eight absA-homologs, and the cutR/S and afsQ1/Q2 two-component regulatory genes involved in secondary metabolite biosynthesis in wild type S. coelicolor. II. Construction of the corresponding isogenic mutant strains for each of the two-component regulators noted in Aim I, for subsequent S. coelicolor genome microarray analysis. Phenotypic profiling (e.g. growth rate, antibiotic biosynthesis, morphological characteristics) will be performed for each isogenic strain. Ill. Construction of recombinant S. coelicolor strains with engineered regulatory gene::gfp fusions to study at the proteomic level temporal and spatial expression patterns for secondary metabolism. From Specific Aims I - Ill, combine Boolean modeling with data from genomic microarray, mutant phenotype profiling and GFP expression analysis to decode the primary network of regulatory circuits in S. coelicolor secondary metabolism. With these methods established, apply high throughput approaches to additional regulatory systems identified using the methods of Aims I -Ill to establish the detailed layered regulatory network involved in control of antibiotic metabolic pathway gene expression in the S. coelicolor genome.